In humans and other multicellular organisms, complex tissues with different cell types must be formed from a uniform population of precursor cells. Cellular differentiation is critical in the development of a mature organism from a single cell, and in generating and replacing differentiated cells from stem cells in adults. Loss of control of differentiation can result in cancer. One pathway by which cell fate is determined is the "Notch" signaling pathway, a cell-cell communication pathway that relays information between neighboring cells (through a cell surface receptor protein called Notch) and processes this information inside the cell to tailor cell fate as needed. We are investigating the quantitative and structural mechanisms of Notch signaling. We wish to understand how Notch pathway proteins inside the signal-receiving cell control fate. We are studying two distinct Notch signaling processes, each controlled by a distinct cellular regulator (Deltex and CSL). Thermodynamic, biochemical, and high resolution structural analysis of the Deltex, CSL, the Notch receptor, and other effectors will be used to advance understanding of the molecular interactions underlying these cell fate decisions. We will determine how Deltex binds Notch, how it modifies Notch (via ubiquitination), and which receptor proteins pair with which Deltex proteins. Studies will reveal how bivalent recognition of CSL by Notch couples to increase binding strength, and how it allosterically turns this transcription factor on, and how a human virus exploits this pathway. We will test the functional significance of our findings through genetic studies in the fruit fly and transcription assays in cultured cells. We will take advantage of a Structural Genomics Consortium pipeline (University of Toronto) to increase the number of Notch proteins available for biophysical and biochemical analysis. Together, these studies will provide an understanding of Notch signaling that connects atomic structure to quantitative mechanism and function in the organism. Our research to better understand Notch signaling is important to human health, since defects in the Notch pathway lead to various forms of human cancer and to developmental malformities, and may ultimately contribute to treatment of these conditions. Our research is also important for understanding stem cells biology, which is maintained by Notch signaling.